A. Unmet Need for Pancreatic Endocrine Cells
Insulin-dependent diabetes is a disease characterized by a loss of the insulin producing cells of the pancreas. The insulin producing cells, also called “beta” cells, normally reside in small spherical structures termed “islets of Langerhans,” which are dispersed throughout the pancreas. It is proven in humans and animals that the transplant of replacement islets of Langerhans, containing functional beta cells, can cure insulin-dependent diabetes. In this procedure, islets are purified from the pancreata of one or more deceased organ donors and injected into one of various sites in the body. Some islets survive the procedure and establish residency in the body where they make and secrete insulin. This can suffice to cure the patient for a few years, until the end of the lifespan of the transplanted islets. See Shapiro (2011) and Robertson (2010).
Although this process effectively treats diabetes, there are insufficient donor pancreata to treat more than a tiny fraction of diabetic patients. For this reason alternative sources of beta cells or pancreatic islets are needed.
B. Alternative Source of Pancreatic Cells
A prospective source of replacement pancreatic cells that has garnered much attention is pluripotent stem cells. Pluripotent stem cells are harvested from an embryo, or they can be created artificially by directing fully differentiated somatic cells to an embryonic-like state; that is, by “reprogramming” adult cells to resemble cells that are harvested from an embryo. Whether by harvesting or reprogramming, all pluripotent stem cells share three characteristics:                Expression of stem cell genes: They express genes typically expressed in the early mammalian embryo.        Immortality: They can be expanded in culture to theoretically unlimited quantities.        Maturation into all lineages of the mammalian body: All adult organs are derived from one of three tissue lineages of the early embryo. These are endoderm, from which the pancreas and other gut organs are formed, mesoderm, from which muscle and skeleton are formed, and ectoderm, from which brain and skin are formed.See Yamanaka (2012), Plath (2011), Lai, (2011), and Stover (2011).        
Protocols have been devised to manipulate pluripotent stem cells to form pancreatic cells. Some of these protocols are capable of driving the differentiation of pluripotent stem cells to a form resembling that of fetal progenitors of the islets of Langerhans. See Kroon (2008), and Rezania (2011). None yields pancreatic cells, however, that are capable of further maturation into functional, insulin-producing cells that could be used therapeutically, in the manner mentioned above.
Conventional protocols for obtaining pancreatic progenitors have a number of drawbacks and disadvantages, but key among them are these:                The populations of pancreatic progenitors are impure, being contaminated by non-pancreatic cells. Pluripotent stem cells are predisposed to form all three germ layers. Therefore, even the most efficient protocol produces populations of pancreatic cells intermixed with non-pancreatic cells.        The populations of pancreatic progenitors are contaminated by immature cells. These cells have retained the property of immortality and can initiate a tumor after transplant to a patient.        The pancreatic progenitors fail to mature into fully functional insulin producing beta cells.        The protocols for culturing the cells employ reagents and procedures that regulatory agencies generally do not accept for human use.See Matveyenko (2010), Tahamtani (2013). See also Title 21, U.S. Code of Federal Regulations, part 1271.C. Cellular Heterogeneity of the Adult Pancreas        
Islets originate during embryogenesis from progenitor cells that bud off from the developing pancreatic ducts. During the life of a healthy individual, beta cells are produced exclusively through replication of existing beta cells. See Dor (2004). The process of beta cell replication occurs more quickly during periods of weight gain, pregnancy, and recovery following pancreatic injury. Isolated beta cells have not replicated in culture heretofore without losing their mature properties. See Pagluca (2013).
Pancreatic stem cells or progenitor cells have not been identified in mature tissue through lineage tracing experiments. Nevertheless, a number of publications have described cells, isolated from the mammalian pancreas, that are said to display some stem cell characteristics. These cells have been identified in the ductal tissue, in the exocrine tissue, and in the islets themselves. For instance, see Gong (2012), Noguchi (2010), and Ciba (2009). They are described as having limited replicative ability and being induced to express insulin.
In addition, a number of published patent documents describe adult stem cells said to be harvested directly from the mature pancreas. See U.S. Pat. No. 6,436,704, U.S. Pat. No. 6,815,203, U.S. Pat. No. 7,544,510, U.S. Pat. No. 8,110,399 and U.S. Pat. No. 8,377,689, and also published U.S. application No. 2004/0115805.
These cell populations have not been manufactured at a scale required to treat a patient, and none has been shown to secrete insulin appropriately in response to glucose. For these reasons, these cells populations have not shown clinical benefit.
For example, U.S. Pat. No. 8,377,689 speaks of pancreatic cells that are said to replicate in culture and to be induced to express insulin. As described, however, these cells had limited replicative ability and did not mature into fully functional beta cells or, at least, were incapable of reversing diabetes in rodent models. That is, the results actually obtained are said to show, in a “diabetic mouse” model, “a recovery from . . . hyperglycemia (>400 mg/dl) to near normal (<300 mg/dl) within five weeks, while the non-transplanted diabetic mouse was hyperglycemic throughout the study period.” Column 45, after line 18 et seq. (emphasis added). In the diabetic mouse model, however, a “normal” is about 150 mg/dl, while a persistent reading above 250 mg/dl, as was reported, is considered proof of a stable diabetic state. See Dang (2013), for example.
Thus, the potential of strategies for curing diabetes by the transplant of surrogate beta cells or islets of Langerhans has gone largely unrealized for want of a scalable source of pharmaceutical-grade therapeutic pancreatic cells.
This body of work is distinct from the invention described here because it pertains to the isolation of a stem cell from a mammalian pancreas. This invention pertains to the isolation of a fully mature, non-stem cell from a pancreas or other source that is manipulated in culture to adopt stem cell characteristics. This method relies on harnessing the genetic diversity of mature cells within human organs to identify the subpopulation that can be manipulated to become a therapeutically useful stem cell. The genetic diversity of cells present in mature human tissues has been appreciated only recently, and conventional understanding is incomplete regarding cell heterogeneity within the mature pancreas.